Light-Controlled Cell Factories: Employing Photocaged Isopropyl-β-d-Thiogalactopyranoside for Light-Mediated Optimization of lac Promoter-Based Gene Expression and (+)-Valencene Biosynthesis in Corynebacterium glutamicum

Abstract

Precise control of microbial gene expression resulting in a defined, fast, and homogeneous response is of utmost importance for synthetic bio(techno)logical applications. However, even broadly applied biotechnological workhorses, such as Corynebacterium glutamicum, for which induction of recombinant gene expression commonly relies on the addition of appropriate inducer molecules, perform moderately in this respect. Light offers an alternative to accurately control gene expression, as it allows for simple triggering in a noninvasive fashion with unprecedented spatiotemporal resolution. Thus, optogenetic switches are promising tools to improve the controllability of existing gene expression systems. In this regard, photocaged inducers, whose activities are initially inhibited by light-removable protection groups, represent one of the most valuable photoswitches for microbial gene expression. Here, we report on the evaluation of photocaged isopropyl-β-d-thiogalactopyranoside (IPTG) as a light-responsive control element for the frequently applied tac-based expression module in C. glutamicum In contrast to conventional IPTG, the photocaged inducer mediates a tightly controlled, strong, and homogeneous expression response upon short exposure to UV-A light. To further demonstrate the unique potential of photocaged IPTG for the optimization of production processes in C. glutamicum, the optogenetic switch was finally used to improve biosynthesis of the growth-inhibiting sesquiterpene (+)-valencene, a flavoring agent and aroma compound precursor in food industry. The variation in light intensity as well as the time point of light induction proved crucial for efficient production of this toxic compound.

Importance: Optogenetic tools are light-responsive modules that allow for a simple triggering of cellular functions with unprecedented spatiotemporal resolution and in a noninvasive fashion. Specifically, light-controlled gene expression exhibits an enormous potential for various synthetic bio(techno)logical purposes. Before our study, poor inducibility, together with phenotypic heterogeneity, was reported for the IPTG-mediated induction of lac-based gene expression in Corynebacterium glutamicum By applying photocaged IPTG as a synthetic inducer, however, these drawbacks could be almost completely abolished. Especially for increasing numbers of parallelized expression cultures, noninvasive and spatiotemporal light induction qualifies for a precise, homogeneous, and thus higher-order control to fully automatize or optimize future biotechnological applications.

FIG 1

Light-controlled gene expression in C. glutamicum using cIPTG as a photoswitch. (A) Two-step release of IPTG from cIPTG by UV-A light-mediated photocleavage and enzymatic hydrolysis of photoproduct esters as described by Young and Deiters (49). (B) Gradual upregulation of EYFP expression in C. glutamicum ATCC 13032(pEKEx2-EYFP) depending on the time of UV-A light exposure (λ_max = 365 nm, 0.9 mW · cm⁻²) using BHI complex (left) or CGXII-glucose minimal medium (right) supplemented with 100 μM cIPTG. Relative EYFP fluorescence values originate from biomass-normalized triplicates and depict low (blue) to high (red) EYFP fluorescence intervals after 20 h of overexpression. Color gradations represent differential expression outputs obtained by variation of induction time or UV-A light exposure. Maximum biomass-normalized fluorescence values obtained in both media are means of triplicates and were arbitrarily set to 100%. hv, light energy.

FIG 2

Comparative analysis of IPTG and cIPTG induction of tac promoter-mediated EYFP expression in C. glutamicum ATCC 13032(pEKEx2-EYFP). Fluorescence ratio intervals of cIPTG- (30 min UV-A, 100 μM) and IPTG-induced (100 μM) EYFP fluorescence are shown during cultivation in BHI complex (A) and CGXII-glucose minimal medium (C) depending on the time of induction and on overexpression times. Fluorescence ratios originate from biomass-normalized triplicates and depict low (blue, superior IPTG induction) to high (red, superior cIPTG induction) ratio intervals in color gradations. Bar plots indicate individual biomass-normalized (norm.) fluorescence values (in arbitrary units [a.u.]) as means of triplicates after 3 h (left) and 20 h (right) of IPTG- (gray) and cIPTG-induced (dark gray) EYFP expression in BHI complex (B) and CGXII-glucose minimal medium (D). Error bars indicate the respective standard deviations.

FIG 3

Flow cytometric single-cell analysis of gene expression induced by IPTG and cIPTG in C. glutamicum ATCC 13032(pEKEx2-EYFP). Distribution of EYFP fluorescence intensities was plotted against the number of cells (counts). Expression cultures were compared in BHI complex (A and B) and CGXII-glucose minimal medium (C and D), and fluorescence was measured after 3 h (A and C) and 20 h (B and D) of EYFP overexpression and induction with IPTG and light after 5 h of cultivation. For light induction, cells were exposed to UV-A light (λ_max = 365 nm) for 10 and 30 min, respectively, with a light intensity of 0.9 mW · cm⁻².

FIG 4

Light-controlled (+)-valencene production in C. glutamicum using CGXII-glucose medium. (A) Biosynthetic route for (+)-valencene production (3) based on the MEP pathway (1) and appropriate isoprenoid pathway gene deletions for improved FPP precursor supply (2). To improve the metabolic flux toward FPP, the heterologous gene ispA and, at a later stage, the endogenous genes dxs and idi, were overexpressed. (B) Screening of engineered C. glutamicum strains VLC3 to VLC6 for (+)-valencene productivity upon IPTG induction (0.1 mM) after different induction time points. (C) (+)-Valencene production upon IPTG induction (0.1 mM) by strain VLC6 grown for 24 h in a flask (light gray) and FlowerPlate (dark gray). (D) (+)-Valencene productivity profiles (left) indicating valencene titer intervals from low (blue) to high (red) in milligrams per liter after 24 h of production with VLC6 using different times of induction as well as different (c)IPTG concentrations (0.1 and 0.25 mM). Light intensities were incremented in a stepwise manner (100% here correlates to 0.9 mW · cm⁻²). The results obtained for induction after 4 h with 0.1 mM cIPTG marked with an asterisk are shown in more detail on the right. Double asterisks indicate control experiments with IPTG induction. All averaged data originated from the results of at least three independent biological triplicates. MEP, methylerythritol phosphate; IPP, isopentenyl pyrophosphate; DMAPP, dimethylallyl pyrophosphate; GPP, geranyl pyrophosphate; FPP, farnesyl pyrophosphate; GGPP, geranylgeranyl pyrophosphate. Error bars indicate the respective standard deviations.